Perturbations in intracellular cholesterol transport result in loss of homeostatic responses that regulate cholesterol synthesis, uptake and esterification. The role of the plasma membrane cholesterol pool (comprising 90% of total cellular cholesterol) on intracellular cholesterol transport was examined in living cultured cells derived from normal patients and those with Niemann Pick type-C (NP-C) disease, a genetic lesion of cholesterol metabolism. Treatment of fibroblasts with hydroxypropyl-b-cyclodextran, selectively removes plasma membrane cholesterol. 3H-cholesterol resident at the plasma membrane and derived by de novo synthesis from 3H-acetate was removed from normal and NPC human fibroblasts with similar kinetics. In contrast, arrival of exogenously derived LDL3H cholesterol, to the plasma membrane from lysosome was slower in NP-C compared to normal fibroblasts. LDL- cholesterol passes through this pathway is impaired in NP-C cells. 58035, a drug that inhibits ACAT ina the endoplasmic reticulum (ER), causes accumulation of LDL-cholesterol in the ER. Although 58035 blocks ACAT, the drug enhances other homeostatic responses whereas, the NP-C mutation inhibits all homeostatic responses. Thus, exogenously derived cholesterol that reaches the ER can signal these cellular responses but cholesterol that accumulates in the proximal, lysosomal-Golgi segment of the pathway can not. The key enzyme in cellular lipolysis, hormone-sensitive lipase (HSL) was present on the surface monolayer of most triacylglycerol containing lipid droplets in adipocytes and steroidogenic cells stimulated with isoproterenol, whereas little HSL was associated with the surface of lipid droplets in unstimulated cells. In contrast, perilipin, which resides on the surface of lipid droplets in unstimulated cells, appears to disperse from the site in stimulated cells. Thus, the hormone induced increased access of intracellular HSL to its triacylglycerol and cholesterol ester substrates may be modulated by translocation of perilipin from the lipid droplet surface. Lipoprotein lipase (LPL), an enzyme required for lipolysis of triacylglycerol in circulating lipoprotein, is made by parenchymal cells and transported to capillaries. Parenchymal cells, derived from combined lipase deficient (cld/cld) mice make an inactive form of LPL. Our studies on cultured brown adipocytes derived from normal and (cld/cld) newborn mice show that mutant cells cannot transport LPL from site of synthesis in ER to Golgi for processing to an active lipase.